High affinity, low molecular weight displacers for oligonucleotide purification

ABSTRACT

A method for purifying oligonucleotides by displacement chromatography on anion-exchange media, using high affinity, low molecular weight (less than about 10000 Da) displacers, is disclosed. Several examples of high affinity, low molecular weight anionic displacers are provided, including polycyclic aromatic compounds having sulfonic acid moieties attached thereon. The efficacy of the technique for high resolution separation of oligonucleotides is demonstrated for an industrial mixture.

STATEMENT AS TO POSSIBLE RIGHTS UNDER FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with support from the National Instituteof Health under Grant No. GM47372. The United States Government may haverights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to displacement chromatography ofoligonucleotides using low molecular weight, high affinity anionicdisplacers.

BACKGROUND OF THE INVENTION

[0003] Oligonucleotides have generated significant interest as drugcandidates for a wide variety of diseases, in particular as antisensetherapeutics and as potent antibiotics. Several antisenseoligonucleotide drugs are currently undergoing human clinical trials,and many others are in a preclinical phase.

[0004] Oligonucleotides are single strands of nucleic acids with DNA orRNA bases, ranging in length from two to 50 bases or nucleotides.Phosphorothicate derivatives of oligonucleotides have also been utilizedbecause of their higher in vivo stability compared to the parentphosphodiester compounds. In the phosphorothioate derivatives, anon-bridging oxygen atom in the phosphodiester backbone is replaced witha sulfur atom. This substitution enhances nuclease resistance and thus,in vivo stability.

[0005] Solid-phase synthesis methods are now available for large-scalepreparation of oligonucleotides. Solid-state synthesis of anoligonucleotide results in a crude product containing not only thedesired full-length (n-length or n-mer) oligonucleotide, but alsomultiple, closely-related deletion or “failure sequences,” primarily oflength n−1. These so-called failure sequences arise by failure to add abase at the necessary position. Such failures or deletions can arise atmultiple positions along the chain. Multiple failure sequences, oflength (n−1, n−2, n−x), are also present for any given length. Inaddition, (n+1)mers may be present. Therefore, purification methods thatoperate on a preparative scale are needed.

[0006] Chromatographic preparation and purification of oligonucleotidescan potentially provide the necessary scale and purity, but can alsopresent unique challenges. First, oligonucleotides exhibit an extremelyhigh binding affinity for-anion-exchange chromatographic resins ascompared to molecules typically encountered in biopharmaceuticalpurification (for example, proteins). Second, the failure sequencespresent are so closely-related to the desired product that thecomponents are difficult to separate. Finally, oligonucleotides exhibitseveral centers of isomerism leading to the possibility of considerableheterogeneity of the mixtures of product and failure sequences.

[0007] A chromatographic system can be operated in one of two majormodes, elution (including linear gradient, step gradient, and isocraticelution) or displacement. The two modes may be distinguished both intheory and in practice. In elution chromatography, a solution of thesample to be purified is applied to a stationary phase, commonly in acolumn. A mobile phase is chosen such that the sample is neitherirreversibly adsorbed nor totally unadsorbed, but rather bindsreversibly. As the mobile phase is caused to flow over the stationaryphase, an equilibrium is established between the mobile phase and thestationary phase whereby components of the sample pass along the columnat speeds which reflects their affinity for the stationary phaserelative to the other components that may occur in the original sample.The differential migration process is outlined schematically in FIG. 1,and a typical chromatogram is shown in FIG. 2. Of particular note is thefact that the eluting solvent front, or zero column volume in isocraticelution, always precedes the sample off the column.

[0008] A modification and extension of isocratic elution chromatographyis found in step gradient chromatography wherein a series of eluants ofvarying composition are passed over the stationary phase.

[0009] In ion-exchange chromatography, step changes in the mobile phasesalt concentration and/or pH are employed to elute- or desorb materialssuch as, for example, proteins.

[0010] A schematic illustrating the operation of a chromatographicsystem in displacement mode is shown in FIG. 3. The column is initiallyequilibrated with a buffer in which most of the components to beseparated have a relatively high affinity for the stationary phase.Following the equilibration step, a feed mixture containing thecomponents to be separated is introduced into the column and is thenfollowed by a constant infusion of the displacer solution. A displaceris selected such that it has a higher affinity for the stationary phasethan any of the feed components. As a result, the displacer caneffectively drive the feed components off the column ahead of its front.Under appropriate conditions, the displacer induces the feed componentsto develop into adjacent “squarewave” zones of highly concentrated,often pure material. The displacer emerges from the column following thezones of purified components. After the breakthrough of the displacerwith the column effluent, the column is regenerated and is ready foranother cycle.

[0011] An important distinction between displacement chromatography andelution chromatography is that in elution chromatography, desorbents,including salts for ion-exchange chromatography, move through the feedzones, while in displacement chromatography, the displacer front alwaysremains behind the adjacent feed zones in the displacement train. Thisdistinction is important because relatively large separation factors aregenerally required to achieve satisfactory resolution in elutionchromatography, while displacement chromatography can potentially purifycomponents from mixtures having low separation factors.

[0012] A key operational feature which distinguishes displacementchromatography from elution chromatography is the-use of a displacermolecule. In elution chromatography, the eluant-usually has a loweraffinity for the stationary phase than any of the components in themixture to be separated, whereas in displacement chromatography, theeluant, which is the displacer, has a higher affinity.

[0013] Displacement chromatography has some particularly advantageouscharacteristics for process scale chromatography of biologicalmacromolecules such as oligonucleotides. First, displacementchromatography can concentrate components from mixtures. By comparison,isocratic elution chromatography results in product dilution duringseparation. Second, displacement chromatography can achieve productseparation and concentration in a single step. Further, since thedisplacement process operates in the nonlinear region of the equilibriumisotherm, high column loadings are possible. This allows for improvedcolumn utilization compared to elution chromatography. Furthermore,displacement chromatography can purify components from mixtures havinglow separation factors, while relatively large separation factors arerequired for satisfactory resolution in desorption chromatography.

[0014] Preparative ion-exchange chromatography operated in thedisplacement mode is, therefore, a potentially attractive method forpurifying oligonucleotides because of the high resolution and highthroughput that can be obtained. However, displacement chromatography,as it is traditionally known, has a number of drawbacks compared toelution chromatography for the purification of oligonucleotides. Two ofthe major problems are difficulty in regeneration of the column and thepresence of displacer in some of the purified fractions.

[0015] Since the displacement process uses a high affinity compound asthe displacer, the time for regeneration and re-equilibration can belong compared to elution chromatography. The second problem, that ofcontamination by the displacer, has arisen because a commoncharacteristic of displacers used in ion-exchange systems has been theirrelatively high molecular weight. Heretofore the art has taught the useof high molecular weight polyelectrolyte displacers on the assumptionthat it is necessary to have a large polyelectrolyte in order to ensurea higher binding coefficient than the biomolecule that is to bedisplaced. The rationale behind such an assumption is that the bindingof a molecule to an adsorbent surface of an ion-exchange stationaryphase is related only to its characteristic charge. Characteristiccharge is the average number of sites of interaction of a solute with astationary phase. High molecular weight displacers exhibit both of thedisadvantages enumerated above: they bind tightly to the stationaryphase and, therefore, require stringent conditions for regenerating thecolumn, and traces of the displacer that may contaminate the productfraction are difficult to remove.

[0016] Low molecular weight displacers for protein separation that donot require extensive regeneration of the column and that can be readilyremoved from the product have been described by Cramer et al. (U.S. Pat.No. 5,606,033, issued Feb. 25, 1997; U.S. Pat. No. 5,478,924, issuedDec. 26, 1995). However, while the low molecular weight displacersidentified so far have been successful in displacing moderately boundbiomolecules such as proteins, they have been unsuccessful in displacingvery highly retained compounds in ion-exchange systems, for example,oligonucleotides. Prior art disclosures relating to purification ofoligonucleotides in ion-exchange systems have described only the use ofrelatively large polyelectrolytes (>40,000 Daltons) as displacers. Forexample, use of displacement chromatography with a high molecular weightpolyelectrolyte displacer, a sulfonated polysaccaride, for thepurification of oligonucleotides has been reported by Gerstner et al.(J. Nucl. Acids Res. 1995, 23, 2292-2299).

[0017] Therefore, there is a need for a separation process ofoligonucleotides having the advantages of displacement chromatography(high resolution and high throughput) while avoiding the disadvantages(difficult column regeneration and contamination of the product with thedisplacer.)

SUMMARY OF THE INVENTION

[0018] In one aspect, the present invention relates to methods forpurifying an oligonucleotide, or several oligonucleotides, comprising:

[0019] (a) loading said oligonucleotide onto an anion-exchange columnhaving a stationary phase; and

[0020] (b) displacing said oligonucleotide from said anion-exchangecolumn by an anionic displacer having a molecular weight of less thanabout 10,000, said anionic displacer having a higher affinity for saidstationary phase of said anion-exchange column than saidoligonucleotide.

[0021] Preferably, the molecular weight of the displacer is less thanabout 5,000. More preferably, the molecular weight of the displacer isless than about 2,500.

[0022] A preferred composition for the displacer is a substituted orunsubstituted aromatic compound having at least one anionic substituent.Another preferred composition for the displacer is a substituted orunsubstituted aliphatic compound having at least one anionicsubstituent. Preferred anionic substitutents are sulfate, sulfonate,phosphate, phosphonate and carboxylate groups. More preferred anionicsubstitutents are sulfonate groups. A preferred aromatic compound is asubstituted or unsubstituted polycyclic aromatic compound having atleast one anionic substituent. Preferred polycyclic aromatic sulfonatesare amaranth and calcion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a schematic representation of isocratic linear elutionchromatography as typically practiced.

[0024]FIG. 2 is a typical HPLC elution chromatogram.

[0025]FIG. 3 is a schematic representation of displacementchromatography.

[0026]FIG. 4 is a dynamic-affinity plot for three displacers, a typical20 mer phosphorothioate oligonucleotide (SEQ ID NO. 2, ISIS-2105), and aprotein.

[0027]FIG. 5 is a displacement chromatogram for SEQ ID NO. 1 (ISIS-2302)purified using amaranth as the displacer (Example 1).

[0028]FIG. 6a is an analytical anion-exchange chromatogram of theoligonucleotide feed shown in FIG. 5.

[0029]FIG. 6b is an analytical anion-exchange chromatogram of one of thepurified fractions from the displacement shown in FIG. 5.

[0030]FIG. 7a is an analytical capillary gel electropherogram of thefeed from the displacement of the oligonucleotide in FIG. 5.

[0031]FIG. 7b is an analytical capillary gel electropherogram of thepurified pool from displacement of the oligonucleotide in FIG. 5.

[0032]FIG. 8 shows an HPLC chromatogram illustrating the effectiveregeneration of the column used in FIG. 5 (Example 2).

[0033]FIG. 9a is an analytical capillary gel electropherogram of thecrude phosphodiester 20 mer SEQ ID NO. 1 (ISIS-2302) containing severallower retained impurities which appear as shoulders on the main productpeak (Example 3).

[0034]FIG. 9b is a displacement chromatogram for the phosphodiesterillustrated in FIG. 9a.

[0035]FIG. 9c is an analytical capillary gel electropherogram of thepurified pool from the displacement of the phosphodiester illustrated inFIG. 9a.

[0036]FIG. 10 is a displacement chromatogram for a phosphorothioate 20mer SEQ ID NO. 2 (ISIS-2105) purified using displacement chromatography(Example 4).

[0037]FIG. 11 is an anion-exchange chromatogram for the feed of thedisplacement chromatography shown in FIG. 10.

[0038]FIG. 12 is an anion-exchange chromatogram of the purified-pool ofthe displacement chromatography shown in FIG. 10.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0039] The method of the present invention relates to separation andpurification of oligonucleotides by ion-exchange chromatography operatedin the displacement mode. A low molecular weight anionic displacerhaving higher affinity for the stationary phase than theoligonucleotides to be separated or purified is utilized. Theoligonucleotide to be purified is dissolved in a solvent and loaded ontoan anion-exchange column having a stationary phase. The oligonucleotideis eluted with an anionic displacer which displaces the oligonucleotidefrom the anion-exchange column.

[0040] In the context of the present invention, low molecular weightmeans a molecular weight of less than 10,000. Preferably, a displacerused in the method of the present invention has a molecular weight ofless than 5,000. More preferably, the displacer has a molecular weightof less than 2,500.

[0041] Affinity of the displacer for the stationary phase of thechromatographic system relative to the oligonucleotides to be separatedor purified is defined with reference to an improved mathematical modelfor displacement chromatography: Steric Mass Action (SMA) ion-exchangemodel. The SMA ion-exchange model is capable of predicting complexbehavior in ion-exchange systems. This model has three soluteparameters: characteristic charge ν is the number of salt counterionsdisplaced by the solute when it binds to the stationary phase surface;steric factor σ is the number of salt counterion sites on the surfacewhich are shielded by the adsorbed solute and hence unavailable forexchange with any other solute molecules; and equilibrium constant κ isthat for the exchange reaction between the salt counterions and thesolute.

[0042] According to the SMA model, the governing parameter thatregulates the ability of one solute to displace another is dynamicaffinity. Dynamic affinity is defined as:$\lambda = ( \frac{K}{\Delta} )^{\frac{1}{v}}$

[0043] wherein λ is the dynamic affinity, Δ is the displacer partitionratio, and κ and ν are as defined above. Δ is equal to Q_(d)/C_(d),where Q_(d) and C_(d) are the displacer concentrations in the stationaryphase and mobile phase, respectively. The value of parameter Δ varieswith the operating conditions for the displacement, which include theconcentrations of displacer and salt.

[0044] Affinity of the displacer for the stationary phase of ananion-exchange system higher than that of the oligonucleotides to beseparated or purified for the same stationary phase is defined withreference to the SMA model. Higher affinity means that the displacer hasa greater dynamic affinity λ than that of the oligonucleotides. Dynamicaffinity of the displacer and the oligonucleotides can be readilydetermined by constructing a plot of log κ versus ν for the displacerand oligonucleotides. This is call-ed a dynamic affinity plot. The slopeof the line is the dynamic affinity λ of the compound or mixture ofcompounds. Where the line for the displacer falls above orcounterclockwise from the line for the oligonucleotides, the displacerhas higher affinity for the stationary phase under those operatingconditions than does the oligonucleotide or mixture of oligonucleotides.The plot is, therefore, used to determine the ability of a displacer todisplace a given oligonucleotide under the operating conditions for thedisplacement.

[0045] An exemplary dynamic affinity plot is shown in FIG. 4. The valuesof log κ and ν are plotted for a typical phosphorothioate antisenseoligonucleotide, a typical protein and several potential displacers.Viewed in a counterclockwise direction, that is, in order of increasingslopes, starting with the line for a typical protein, this plot showsincreasing affinity of the solutes under the experimental conditions. Itis evident that even the highest affinity low molecular weight displacerpreviously identified for anion-exchange, sucrose octasulfate, did notpossess enough affinity to displace this oligonucleotide. In fact, theoligonucleotide has an equilibrium constant that is several orders ofmagnitude higher than that of a typical anionic protein, β-lactoglobulinA, as shown in FIG. 4.

[0046] The three SMA parameters, ν, σ, and κ, may be determinedexperimentally. The characteristic charge and equilibrium constant ofthe oligonucleotides to be purified may be determined using lineargradient elution retention data with different slopes of the lineargradient (Shukla et al., Ind. Eng. Chem. Res., 1998, 37, 4090-4098,which is herein incorporated by reference). The steric factor of theoligonucleotides may then be determined from the capacities obtainedfrom frontal experiments at two or more displacer concentrations (Gadamet al, J. Chromatogr., 1993, 630, 37-52), which is herein incorporatedby reference). The magnitude of the induced gradient obtained duringthese frontal experiments can provide an independent measure for thecharacteristic charge ν.

[0047] A slightly different procedure may also be employed to obtain theSMA parameters of the displacer. Isocratic experiments may be conductedat different mobile phase salt concentrations, and a plot of log κ′ vs.log (salt concentration), where κ′ is the dimensionless retention timeof a solute at a specific mobile phase salt concentration, may be made.The values of the slope and intercept of the line are used to calculatethe characteristic charge ν and the equilibrium constant κ, respectively(Gadam et al., J. Chromatogr., 1993, 630, 37-52). The frontalexperiments described above may be used to determine the steric factorand an independent measure of ν.

[0048] The retention process in ion-exchange is not solely determined byelectrostatic interactions but may be greatly enhanced by non-specificinteractions. One of the dominant factors which can play a major role ingoverning retention on an anion-exchange resin are hydrophobicinteractions. For example, aromatic compounds having multiple sulfonicacid functionalities may be effective displacers for oligonucleotides.

[0049] Therefore, an anionic displacer having higher affinity for astationary phase of an anion-exchange system may be an aromatic,substituted aromatic aliphatic or substituted aliphatic compoundcontaining at least one anionic substituent. Preferred anionicsubstituents are sulfate, sulfonate, phosphate, phosphonate andcarboxylate groups. More preferred is a substituted or unsubstitutedaromatic anionic displacer containing at least one sulfonate group. Theanionic displacer may also be a polycyclic aromatic or substitutedpolycyclic aromatic compound containing at least one anionicsubstituent. Preferred anionic substituents are sulfate, sulfonate,phosphate, phosphonate and carboxylate groups. A preferred substitutedor unsubstituted polycyclic aromatic anionic displacer contains at leastone sulfonate group. Examples of polycyclic aromatic sulfonatedisplacers having higher affinity for a stationary phase than anoligonucleotide are amaranth (I) and calcion (II), both available fromAldrich Chemical Company. The structures of I and II are shown below.

[0050] Other exemplary aromatic sulfonates which may exhibit higheraffinity for a stationary phase of an anion-exchange system than anoligonucleotide to be purified under the operating conditions of thesystem are New Coccine, Ponceau S, Ponceau SS, Hydroxy Naphthol Blue,Brilliant Black, Anthraquinone Disulfonic Acid, PotassiumIndigotetrasulfonate, Sulfazo III, Reactive Orange 16, Acid AlizarinViolet N, Acid Black 24, Acid Blue 29, Acid Blue 80, Acid Blue 92, AcidBlue 113, Acid Blue 120, Acid Green 25, Acid Green 27, Acid Orange 8,Acid Orange 51, Acid Orange 63, Acid Orange 74, Acid Red 1, Acid Red 4,Acid Red 8, Acid Red 97, Acid Red 106, Acid Red 114, Acid Red 151, AcidRed183, Acid Violet 5, Acid Violet 7, Acid Yellow 17, Acid Yellow 25,Acid Yellow 29, Acid Yellow 34, Acid Yellow 38, Acid Yellow 40, AcidYellow 42, Acid Yellow 65, Acid Yellow 76, and Acid Yellow 99. All ofthese compounds are commercially available from Aldrich ChemicalCompany.

[0051] The method of the present invention may be used with conventionalanion-exchange systems and conventional displacement chromatographyprocedures. An example of a useful anion-exchange column is a Poros HQ/Mcolumn, which has a stationary phase composed of a rigidpolystyrene-divinylbenzene bead covered with a hydrophilic layer. Thecolumn is available from PerSeptive Biosystems, Inc. Displacementchromatography operations are typically carried out by initiallyequilibrating the column with carrier solution and then sequentiallyperfusing with feed, displacer, and regenerant solutions. The feed andthe displacer solutions are commonly prepared in the same buffersolution as the carrier.

[0052] As used herein, the term oligonucleotide includes oligomerscontaining two or more nucleoside subunits having phosphorusinternucleoside linking moieties. Nucleoside subunits according to theinvention have a ribofuranose moiety attached to a nucleobase through aglycosyl bond. oligonucleotides according to the present inventionpreferably comprise from about 5 to about 50 nucleosides. It ismore-preferred that such compounds comprise from about 8 to about 30nucleosides. It is most preferred that such compounds comprise fromabout 15 to about 25 nucleosides.

[0053] Additional objects, advantages, and novel features of the presentinvention will become apparent to those skilled in the art uponexamination of the following examples. The following examples illustratethe invention and are not intended to limit the same. Those skilled inthe art will recognize, or be able to ascertain through routineexperimentation, numerous equivalents to the specific substances,compositions, and procedures described herein. Such equivalents areconsidered to be within the scope of the present invention.

EXAMPLES Example 1

[0054] Displacement Chromatography of a Phosphorothioate 20 mer, SEQ IDNO: 1 (ISIS-2302) GCC CAA GCT GGC ATC CGT CA (P=S)

[0055]FIG. 4 shows a Dynamic Affinity plot for preferred displacers fora typical phosphorothioate oligonucleotide, amaranth (I) and calcion(II), in comparison to a disaccaride bearing eight sulfate groups,sucrose octasulfate. The line for sucrose octasulfate falls below thatof the oligonucleotide, while those for amaranth and calcion fallcounterclockwise (above) to that of the oligonucleotide. Therefore, bothamaranth and calcion have higher affinity for the stationary phase thanthe oligonucleotide.

[0056] The ability of the dynamic affinity parameter to predictreal-world behavior was confirmed for amaranth in a separation of atypical phosphorothioate oligonucleotide. FIG. 5 shows a histogram of adisplacement separation of SEQ ID NO: 1, a 20-mer phosphorothioateantisense oligonucleotide, using amaranth as a displacer on a Poros HQ/Mcolumn (4.6×100 mm I.D., 20 μm particles), at a flow rate of 0.2 mL/min.The carrier solution and mobile phase was a 20 mM NaOH/500 mM NaClsolution. The feed and displacer solution were prepared from the carriersolution and the feed consisted of 11.98 mg oligonucleotide. Aneffective displacement was demonstrated with a sharp boundary betweenzones containing the impurities and the desired oligonucleotide, andbetween zones containing the oligonucleotide and the amaranth displacer.Thus, the histogram demonstrates the resolving power of amaranth indisplacement chromatography of oligonucleotides.

[0057] High temperature anion-exchange analysis of the feed and of thefraction containing the desired oligonucleotide confirmed the purity ofthe product oligonucleotide. Chromatograms from the displacementexperiment show the oligonucleotide fraction collected (FIG. 6a) and thefeed component from the displacement experiment (FIG. 6b). Even thoughthe feed was only about 58% pure, the oligonucleotide fraction had apurity of about 99% by anion-exchange analysis. Capillaryelectropherograms for the feed and product pool from the example in FIG.5 are shown in FIGS. 7a and 7 b.

[0058] The displacement histogram depicted in FIG. 5 was prepared usingthe separation parameters listed above and the general procedureillustrated below. One fractions per minute was collected and analyzedby analytical anion-exchange assay to determine the concentration of thecomponents and their purities. Standard curves for the peak area versusthe oligonucleotide concentration and the peak area versus the displacerconcentration were generated using the sample of pure oligonucleotideand a sample of known concentration for the displacer, respectively. Theconcentration of oligonucleotide and amaranth in each of the fractionswas then determined by using this standard curve to calculate a value ofthe extinction coefficient (peak area per unit concentration). The sameextinction coefficient was assumed for the mono-phosphodiester (P=O)1component and other impurity species since these are chemically verysimilar. For these experiments the cumulative impurity concentration wasdetermined by subtracting the main peak, i.e. the all phosphorothioate(P=S) parent peak, concentration from the total oligonucleotideconcentration in each fraction. A histogram was generated to representthis experimental data by plotting the cumulative concentration ofimpurities, product oligonucleotide and amaranth for each of thefractions vs. volume of column effluent.

Example 2

[0059] Column Regeneration Following Displacement Chromatography

[0060] Following purification of the phosphorothioate oligonucleotide inExample 1 above the column was regenerated. Demonstrating effectiveregeneration of the stationary phase after a displacement run isimportant to enable the use of a compound as an efficient displacer.FIG. 8 shows an HPLC chromatogram with the breakthrough curves fornitrate ions before and after the column had been put through severalsuccessive displacement steps as illustrated in Example 1. As can beseen, the breakthrough curves overlay each other signifying an effectivecolumn regeneration using standard protocols. The 4 step regenerationprotocol is shown below.

[0061] 1) Elute column with 5 column volumes of 2.5 M NaCl and 20 mMNaOH.

[0062] 2) Elute column with 5 column volumes water.

[0063] 3) Elute column with 5 column volumes 25% v/v acetonitrile and 20mM NaOH.

[0064] 4) Elute column with 5 column volumes water.

Example 3 Displacement Chromatography of a Phosphodiester 20 mer, SEQ IDNO: 1 (ISIS-2302) GCC CAA GCT GGC ATC CGT CA (P=O)

[0065] The column that was regenerated after about 3 displacement runsin Example 2 was used to purify SEQ ID NO: 1 as a Phosphodiester. Thegeneral procedure illustrated in Example 1 was followed. Hightemperature anion-exchange analysis of the feed mixture for thisseparation is shown in FIG. 9a. The mixture contained several lowerretained impurities which appear as shoulders on the main product peak.The displacement chromatogram resulting from the displacement of thisoligonucleotide using amaranth is shown in FIG. 9B. Again, an effectiveseparation was achieved resulting in a yield of 69.7% at 99% purity bythe anion-exchange assay. The analytical capillary gel electropherogramof the purified pool of the purified phosphodiester is shown in FIG. 9c.

Example 4 Purification of a Phosphorothioate 20 mer Using DisplacementChromatography, SEQ ID NO: 2 (ISIS-2105) TTG CTT CCA TCT TCC TCG TC(P=S)

[0066] SEQ ID NO: 2, a phosphorothioate 20 mer, was purified bydisplacement chromatography using amaranth as the displacer. Thechromatography was performed as per the procedure illustrated in Example1 above. Column: 4.6×100 mm, Poros HQ/M; mobile phase: 20 mM NaOH/500 mMNaCl; loading: 10 mg phosphorothioate oligonucleotide; flow rate: 0.2mL/min; feed purity=91.22%. FIG. 10 shows the displacement chromatogram.FIG. 11 shows the anion-exchange chromatogram and FIG. 12 shows theanion-exchange chromatogram of the purified pool.

[0067] While the invention has been particularly shown and describedwith reference to the preferred embodiments thereof, it will beunderstood by those skilled in the art that other changes in form anddetails may be made without departing from the spirit and scope of theinvention.

What is claimed is:
 1. A method for purifying an oligonucleotidecomprising: (a) loading said oligonucleotide onto the stationary phaseof an anion-exchange column; and (b) displacing said oligonucleotidefrom said stationary phase by an anionic displacer having a molecularweight of less than about 10,000, said anionic displacer having a higheraffinity for said stationary phase than said oligonucleotide.
 2. Themethod of claim 1 wherein said anionic displacer has a molecular weightof less than about 5,000.
 3. The method of claim 2 wherein said anionicdisplacer has a molecular weight of less than about 2,500.
 4. The methodof claim 1 wherein said anionic displacer is a substituted orunsubstituted aromatic compound having at least one anionic substituent.5. The method of claim 4 wherein said anionic substituent is selectedfrom the group consisting of sulfate, sulfonate, phosphate, phosphonateand carboxylate groups.
 6. The method of claim 5 wherein said anionicsubstituent is a sulfonate group.
 7. The method of claim 4 wherein saidanionic displacer is a substituted or unsubstituted polycyclic aromaticcompound having at least one anionic substituent.
 8. The method of claim7 wherein said anionic substituent is selected from the group consistingof sulfate, sulfonate, phosphate, phosphonate and carboxylate groups. 9.The method of claim 8 wherein said anionic substituent is a sulfonategroup.
 10. The method of claim 9 wherein said displacer is amaranth. 11.The method of claim 9 wherein said displacer is calcion.
 12. The methodof claim 1 wherein said anionic displacer is a substituted orunsubstituted aliphatic compound having at least one anionicsubstituent.
 13. The method of claim 12 wherein said anionic substituentis selected from the group consisting of sulfate, sulfonate, phosphate,phosphonate and carboxylate groups.
 14. The method of claim 13 whereinsaid anionic substituent is a sulfonate group.
 15. The method of claim 1wherein said oligonucleotide comprises from 10 to 40 nucleotide bases.16. The method of claim 1 further comprising regenerating saidanion-exchange system by perfusing with a regenerant solution.
 17. Amethod for purifying an oligonucleotide comprising: (a) loading saidoligonucleotide in a suitable mobile phase on a stationary phase of ananion-exchange system; and (b) displacing said oligonucleotide from saidanion-exchange system by elution with a polycyclic aromatic sulfonatedisplacer.
 18. The method of claim 17 wherein said polycyclic aromaticsulfonate displacer comprises amaranth.
 19. The method of claim 17wherein said polycyclic aromatic sulfonate displacer comprises calcion.